Hypersensitivity in human beings to certain substances in the environment may manifest itself in the form of several different disease symptoms. Of these, the syndromes of allergic bronchial asthma, neurodermatitis, vasomotor rhinitis, hay fever (or pollinosis) and food allergies are among the most widely known. Despite the fact that in any human population in any geographical location the exposure to the inciting agents (or "allergens") is about equal for each individual, it is nevertheless a fairly constant proportion of about 10% of any given population that really develops manifest disease symptoms. This polar group of the human population is identified as being "atopic", meaning that the afflicted individuals have a genetically determines predisposition to respond with disease symptoms to "allergens" in the environment, in contrast to the non-atopic population which responds only at much higher levels of exposure, or not at all. Current scientific theory holds that manifest allergic disease in atopic subjects is caused by the interaction of such environmental allergens with specific antibodies, so-called immunoglobulins of the IgE-isotype, which recognize the allergen as an individual macromolecular entity or, in current terminology, as an "antigen". Since this IgE-class antibody is indeed predominantly found in the blood and tissues of atopic people, the inherited physiological condition of "atopy" is considered to be primarily an inborn abnormality of the immunological defense system, causing atopic people to be "high IgE-responders".
The medical treatment of human disease normally aims at combatting the ultimate physiological effects of the chain-reactions initiated by the allergen-IgE interaction, and therefore relies on antihistaminic drugs, cholinergic antagonists or adrenergic agonists, on repeated injections with the offending allergen to induce a shift of the antibody response in the direction of presumed "protective" IgG-antibodies and, prophylactically, on identifying and eliminating the source of the particular allergen(s) in the patient's environment. The science and technology of identifying and quantifying the causative allergens in a patient's direct environment, i.e. in the home or workplace, has remained underdeveloped. Further technologies in this field are badly needed in order to provide each individual patient or group of patients with sound advice on "sanitation" of their environment and reduction of the allergen load, prior to taking drugs for alleviating the symptoms. Such measures depend on the techniques available for allergen detection, and the development of these derives from current scientific insight into their nature and mode(s) of action.
The current viewpoint of human allergic disease of the so-called "immediate-type" (Type I in immunological classification) hinges on the structural concept of allergens being recognized as molecular entities (or as "antigens") by specific IgE-antibodies. The clinical detection and identification of allergens by their capacity to induce strong wheal and flare (urticarial and erythemal) reactions when injected into the skin of specifically sensitized atopic patients for obvious reasons is too cumbersome to allow adequate monitoring of the allergenic potential of the environment. Laboratory methods developed for this purpose have therefore relied on elaborate and expensive immunochemical techniques for the identification and quantification of allergens in extracts of environmental substrates by means of binding studies to IgE-antibodies in individual or pooled human blood serum samples from allergic individuals.
The major allergenic substances for atopic people derive from components in the dust accumulating in houses. Many different organisms contribute to this allergen load, in particular mites and other arthropods, insects, yeasts and moulds and, in the relevant seasons, wind-distributed pollen grains from grasses, trees and weeds from the outside environment. In some dwellings, allergens shed with the skin scales, dried saliva or urinary constituents from animal pets like cats, dogs, birds and other animals may contribute substantially to the allergen load in an individual's personal environment. In order to identify each and every one of these different allergens by techniques based on the binding of specific IgE-antibodies, a very large variety of blood serum samples of allergic individuals would have to be available. This particular prerequisite will continue to hamper the development of adequate control measures of the allergic environment of man.
In an attempt to quantify the contribution of the so-called house dust mites Dermatophagoides pteronyssinus and D. farinae, which live in house dust and excrete highly potent inhalant allergens for man, it has been proposed to quantify the nitrogenous excretion product guanine in samples taken from the dust. A technological procedure has been made available based on this principle and has been described in the scientific and patent literature EP 0 144 820, 0 152 068 and 0 174 448. Bischoff E. Schirmacher W: Allergologie 1984; 7: 446-9; 1985; 8: 36-8; Bischoff E. Schirmacher W, Schober G: Allergologie 1985; 8: 97-9. Bronswijk JEMH van, Bischoff E, Schirmacher W, Berrens L, Schober G, J Med Entomol 1986; 23: 217-8). However, the results obtained do not really provide data of relevance to the desired estimation of the environmental allergenic load associated with manifest allergic disease. The nitrogenous waste product guanine produced by arthropods, spiders, insects, birds and some mammals has shown to be a highly insoluble low-molecular weight organic compound totally devoid of any detectable allergenic activity. The measurement of its contribution to the dry weight of house dust samples can therefore be no more than an approximate index of the total biomass of organisms contributing to the overall pool of components in house dust, whether allergenic or not. Results obtained in this fashion can, therefore, not be extrapolated or converted into any realistic parameter for the environmental content of biologically active allergenic macromolecules deriving from a great many potentially allergenic substrates. In accordance, investigations into the relationship between guanine content of house dust samples and immunological parameters for true allergen content have shown only crude statistical approximations of the presumed past and present population of mites in the dust samples {Bronswijk JEMH van. Exp Appl Acarology 1986; 2: 231-8.; Bronswijk JEMH van, Reumer JWF, Pickard R. Exp Appl Acarology 1987; 3: 271-8.; Pauli G, Hoyet C, Tenabene A, Le Mao J, Thierry R, Bessot JC. Clin Allergy 1988; 18: 383-92}. The availability of a method for the estimation of the total or specific allergen content of environmental dust samples based on the measurement of biologically really relevant allergenic properties and not on biologically inert and accidental by-products would be desirable.
In contrast to the commonly accepted structural theory of allergy, whereby allergenic macromolecules are recognized as antigens by specific (IgE-) antibodies, it has been proposed that allergens may by themselves be biologically active molecules, exerting their action in atopic people by an intrinsic molecular property that distinguishes them from all other antigens (Berrens L. Ann Allergy 1976; 36: 351-61). For the deployment of this inherent biological activity in allergic subjects no antibodies of any class have been considered essential. In this connection, it has for example been demonstrated that the collection of allergens in house dust exerts the function of activating the so-called complement system of zymogens in human beings without the detectable participation of antibodies {Berrens L, Guikers CLH, Van Rijswijk-Verbeek J. Immunochemistry 1976; 13: 367-72).
According to the present invention it was found that major allergens are proteolytic enzymes. This is totally unexpected in view of the scientific literature on possible enzymes in house dust or dust mite extracts proved highly discouraging (Bousquet J, Hale R, Guerin B, Michel F-B. Ann Allergy 1980; 45: 316-21; Stewart GA, Butcher A. Lees K, Ackland J. J Allergy Clin Immunol 1986; 77: 14-24): no enzymes even remotely capable of initiating enzyme cascades were detected.